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Bacterial Polysaccharides pp 245–253Cite as

In Vitro Characterization of a Multidomain Glycosyltransferase Using Fluorescently Tagged Synthetic Acceptors

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1954))

Abstract

In vitro assays using fluorescently tagged sugar residues can facilitate the characterization of glycosyltransferase function. Here we describe the use of in vitro assays to characterize the three glycosyltransferase modules of the protein designated WbbB from Klebsiella pneumoniae O12. This protein combines key activities necessary to synthesize the O antigenic polysaccharide portion of lipopolysaccharide. The specificities of the three glycosyltransferases were investigated in vitro, using purified proteins, the activated donor sugars (dTDP-Rha, UDP-GlcNAc and CMP-β-Kdo) and synthetic acceptors terminating in either α1,3-linked Rha or β1,4-linked GlcNAc. The reaction products were verified by mass spectrometry and nuclear magnetic resonance methods.

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References

  1. Clarke BR, Richards MR, Greenfield LK et al (2011) In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan. J Biol Chem 286:41391–41401

    Article  CAS  Google Scholar 

  2. Ovchinnikova OG, Mallette E, Koizumi A et al (2016) Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99). Proc Natl Acad Sci U S A 113:3120–3129

    Article  Google Scholar 

  3. Williams DM, Ovchinnikova OG, Koizumi A et al (2017) Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control. Proc Natl Acad Sci U S A 114:E1215–E1223

    Article  CAS  Google Scholar 

  4. Raetz CRH, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700

    Article  CAS  Google Scholar 

  5. Whitfield C, Trent MS (2014) Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem 83:99–128

    Article  CAS  Google Scholar 

  6. Cress BF, Englaender JA, He W et al (2014) Masquerading microbial pathogens: capsular polysaccharides mimic host-tissue molecules. FEMS Microbiol Rev 38:660–697

    Article  CAS  Google Scholar 

  7. Lerouge I, Vanderleyden J (2002) O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. FEMS Microbiol Rev 26:17–47

    Article  CAS  Google Scholar 

  8. Vinogradov E, Frirdich E, MacLean LL et al (2002) Structures of lipopolysaccharides from Klebsiella pneumoniae. Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains. J Biol Chem 277:25070–25081

    Article  CAS  Google Scholar 

  9. Lombard V, Golaconda Ramulu H, Drula E et al (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:490–495

    Article  Google Scholar 

  10. Graninger M, Nidetzky B, Heinrichs DE et al (1999) Characterization of dTDP-4-dehydrorhamnose 3,5-Epimerase and dTDP-4-dehydrorhamnose Reductase, Required for dTDP-L-rhamnose Biosynthesis in Salmonella enterica Serovar Typhimurium LT2*. J Biol Chem 274:25069–25077

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Akihiko Koizumi, Iain L. Mainprize, Matthew S. Kimber, and Todd L. Lowary for their contributions to the projects originally published as refs. 2 and 3. This work was supported by the Natural Sciences and Engineering Research Council of Canada. C.W. acknowledges additional support from the Canada Research Chairs program.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

    Danielle M. Williams, Olga G. Ovchinnikova & Chris Whitfield

Authors
  1. Danielle M. Williams
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  2. Olga G. Ovchinnikova
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  3. Chris Whitfield
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Corresponding author

Correspondence to Chris Whitfield .

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Editors and Affiliations

  1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Inka Brockhausen

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Williams, D.M., Ovchinnikova, O.G., Whitfield, C. (2019). In Vitro Characterization of a Multidomain Glycosyltransferase Using Fluorescently Tagged Synthetic Acceptors. In: Brockhausen, I. (eds) Bacterial Polysaccharides. Methods in Molecular Biology, vol 1954. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9154-9_19

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  • DOI: https://doi.org/10.1007/978-1-4939-9154-9_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9153-2

  • Online ISBN: 978-1-4939-9154-9

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